Since the discovery that sera of patients with systemic lupus erythematosus (SLE) contain antibodies to nuclear components, more refined methods have been developed to define the antigens involved in the reaction and to measure the concentration of antibody. Methods used to establish the nature of the antigen and antibody have included complement fixation, hemagglutination, precipitation of soluble immune complexes with ammonium sulfate, immunoprecipitation by double diffusion in agar and immunohistology. The reaction has been quantitated by dilution of the sera for immunohistology, by the extent of binding of DNA by the sera, and by solid phase radio immunoassay. The use of antibodies coupled to enzymes has opened the possibility of using such conjugates to replace the radioactive label in immunoassay systems.
Pesce et al (Clinical Chem. 20/3, 353-359, 1974) described a technique consisting of preparing an antigen on an insoluble support, layering the test serum with anti-DNA, and using this serum as an antigen to react with an anti-human IgG labeled with enzyme-peroxidase. In principle, the amount of IgG bound to the insoluble support is proportional to the amount of antibody contained in the serum. Pesce et al demonstrated that a solid phase noncompetitive binding assay, with use of a second antibody conjugated to an enzyme as the quantitive or detector system, is a valid way to measure antibody to DNA in serum.
Patients with systemic autoimmune diseases produce a variety of antibodies directed against normal cellular components. Several well characterized autoantibody systems are directed against specific proteins or nucleic acid-protein complexes (Tan, E. M (1989) Adv. Immunol. 44, 93-151). Sera from patients with connective tissue diseases often contain antibodies against cellular components consisting of proteins associated with small RNA molecules of 80-200 nucleotides in length. Autoantibodies against the nuclear type of these small RNA-protein complexes (snRNP) are mostly found in sera from patients with SLE or SLE overlap syndromes. Several specificities have already been described. There are antibodies that precipitate (U1)snRNP only, due to the fact that they recognise one or more of the (U1)snRNP specific proteins viz. 70K, A and C. Another specificity, anti-Sm, precipitates all the major nucleoplasmic snRNPS due to the fact that these antibodies recognise the proteins B'/B and D which are common to these snRNPS (v. Venrooij and Sillekens, Clin. Exp. Rheum., 1989).
The antibodies in the patients sera are in all these cases directed against proteins contained in the snRNP complexes. Although nucleic acids are generally poor immunogens, autoantibodies directed against nucleic acids have been reported. The majority of studies of nucleic acid antibodies have concerned anti-DNA activities, although examples of anti-viral RNA autoantibodies have been described.
Antibodies against the (U)RNA components of snRNP are thought to be rare (Tan, 1989). Wilusz and Keene described in 1986 (J. Biol. Chem, 261, 5467-5472) the presence of anti-(U1)RNA autoantibodies in two anti-RNP sera. From this article no general conclusions regarding an association between anti-RNP and/or Sm activity and anti-(U1)RNA activity could be drawn. Deutscher and Keene described in 1988 (P.N.A.S., 85, 3299-3303) that the part of the (U1)RNA recognised by one of said sera was mapped and found to include the second stem-loop of the RNA.
We initiated a study in which a large number of anti-snRNP sera were tested for the presence of anti(U1)RNA antibodies. Via screening with CIE (counter immuno-electrophoresis) and IB (immunoblotting), 118 sera containing anti-snRNP activity were identified. Following RNP immunoprecipitation sixty sera were classified as anti-(U1)RNP and 18 sera as anti-Sm. Twenty-five sera contained anti-RNP as well as anti-Sm antibodies while 15 sera exhibited anti-(U1,U2)RNP activity.
These 118 sera were all tested for their ability to precipitate de-proteinized (U)RNA's. For this purpose total 32P labeled Hela cell RNA, purified by extensive pronase and phenol/detergent treatment, but also in vitro synthesized (U)RNA's, was used as antigen in the precipitation assay. Immunoprecipitation was carried out in IPP/500 mM NaCl, 10 mM Tris pH 7,5 and 0.05% Nonidet P40.sup.(R)). The overall results show that 45 out of the 118 sera (about 40%) contained anti-(U1)snRNA antibodies. These antibodies were not present in the anti-Sm sera. The autoantibodies were always directed against (U1)snRNA and are most often present in patients with SLE overlap syndromes. Using immunoprecipitation under stringent conditions (500 mM NaCl) with in vitro synthesized (U1)RNA or in vivo labeled total Hela cell RNA, it has been found that about 65% of the SLE overlap syndrome and about 30% of the SLE sera with anti-snRNP activity contained antibody against (U1)RNA. No autoantibodies against other (U)RNAs were detected. Using in vitro synthesized stem-loops of (U1)RNA, it is possible to detect antibodies against all individual stem-loops of (U1)RNA. The major antigenic regions are however located in the so-called second (the B) and fourth (the E) stem-loop domain of (U1)RNA. It appears that the nucleotide sequence UUCG (nrs. 150-154) in the fourth E-stem loop is extremely essential as recognition site by patient specific antibodies.
The correlation between anti-(U1)RNA antibody level and autoimmune disease activity was most surprising.